U.S. patent application number 10/511413 was filed with the patent office on 2005-11-17 for system for enhanced targeted delivery.
This patent application is currently assigned to President and Fellows of Harvard College. Invention is credited to Deaver, Daniel R., Edwards, David A., Fulford-Jones, Thaddeus R.F., Kucera, Jane N., Tee, Shang-You.
Application Number | 20050255152 10/511413 |
Document ID | / |
Family ID | 29251103 |
Filed Date | 2005-11-17 |
United States Patent
Application |
20050255152 |
Kind Code |
A1 |
Edwards, David A. ; et
al. |
November 17, 2005 |
System for enhanced targeted delivery
Abstract
A method and compositions for targeted drug delivery have been
developed. The compositions include a targeting molecules such as a
hormone that specifically binds to a receptor on the surface of the
targeted cells; a drug to be delivered, such as a toxin that will
kill the targeted cells; and a nanoparticle, which contains on or
within the nanoparticle, the drug to be delivered, as well as has
attached thereto, the targeting molecule. Nanoparticles can consist
of drug or drug associated with carrier, such as a controlled or
sustained release materials like a poly(lactide-co-glycolide), a
liposome or surfactant. The compositions are administered by
injections in most cases, although compositions can be applied
topically orally, nasally, vaginally, rectally, and ocularly.
Compositions can also be administered to the pulmonary or
respiratory system, most preferably in an aerosol.
Inventors: |
Edwards, David A.; (Boston,
MA) ; Deaver, Daniel R.; (Franklin, MA) ;
Fulford-Jones, Thaddeus R.F.; (Cambridge, MA) ;
Kucera, Jane N.; (Acton, MA) ; Tee, Shang-You;
(Allston, MA) |
Correspondence
Address: |
PATREA L. PABST
PABST PATENT GROUP LLP
400 COLONY SQUARE
SUITE 1200
ATLANTA
GA
30361
US
|
Assignee: |
President and Fellows of Harvard
College
|
Family ID: |
29251103 |
Appl. No.: |
10/511413 |
Filed: |
May 9, 2005 |
PCT Filed: |
April 17, 2003 |
PCT NO: |
PCT/US03/12261 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60373915 |
Apr 18, 2002 |
|
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Current U.S.
Class: |
424/450 ;
424/489; 514/1.2; 514/10.3; 514/9.8 |
Current CPC
Class: |
A61K 9/1271 20130101;
A61K 9/127 20130101; B82Y 5/00 20130101; A61K 31/704 20130101; A61K
47/62 20170801; A61K 47/6933 20170801; A61K 9/1278 20130101 |
Class at
Publication: |
424/450 ;
514/012; 424/489 |
International
Class: |
A61K 038/22; A61K
009/127; A61K 009/14 |
Claims
1. A composition for delivery of a therapeutic, prophylactic or
diagnostic agent comprising: a nanoparticulate carrier comprising
therapeutic, prophylactic or diagnostic agent to be delivered, and
a targeting molecule bound to the surface of the carrier, wherein
the carrier is targeted to a specific population of cells where the
agent is to be delivered.
2. The composition of claim 1 wherein the carrier is selected from
the group consisting of liposomes and microparticles.
3. The composition of claim 2 wherein the microparticles are formed
of the agent to be delivered.
4. The composition of claim 2 wherein the microparticles are formed
of a polymeric material.
5. The composition of claim 1 wherein the agent to be delivered is
a cytotoxic compound.
6. The composition of claim 1 wherein the targeting molecule is a
hormone.
7. The composition of claim 1 further comprising a gel enhancing
uptake of the carrier into a cell.
8. A method of delivering a therapeutic, prophylactic or diagnostic
agent comprising administering to a patient or tissue a composition
comprising a nanoparticulate carrier comprising therapeutic,
prophylactic or diagnostic agent to be delivered, and a targeting
molecule bound to the surface of the carrier, wherein the carrier
is targeted to a specific population of cells where the agent is to
be delivered.
9. The method of claim 8 wherein the composition is administered to
sterilize an animal.
10. The method of claim 8 wherein the composition is administered
to treat cancer or endometriosis.
11. The method of claim 8 wherein the composition is administered
by injection.
12. The method of claim 8 wherein the composition is administered
by pulmonary or intranasal or intravaginal or intrarectal delivery.
Description
FIELD OF THE INVENTION
[0001] The present invention is generally in the field of targeted
drug delivery, especially hormone targeted cytotoxic drugs in a
microparticulate formulation, optionally in combination with a gel
enhancing cell permeation. In one embodiment, the system consists
of a simple and quick injection that causes immediate, permanent
sterilization of an animal.
[0002] This application claims priority to U.S. Ser. No. 60/373,915
"System for Enhanced Targeted Delivery" Filed Apr. 18, 2002, by
David A. Edwards.
BACKGROUND OF THE INVENTION
[0003] Currently, the main method of spaying and neutering is
surgical ovariectomies and orchiectomies. This method has a number
of drawbacks. First, it is a time intensive and labor-intensive
procedure, especially in the case of ovariectomies, which are
understandably more invasive. Second, the surgical procedures are
relatively costly. According to figures from the American Humane
Society, the national average cost for cat castration is about $65
while for dogs the average is approximately $140. Lastly, many pet
owners fail to get their pets fixed because of the violent nature
of the surgical solution. Owners feel that their relationship with
the pet will be adversely affected by the operation.
[0004] The American Veterinary Medical Association (AVMA) reports
about 62 MM cats and 53 MM dogs nationwide with 77% of the cats and
50% of the dogs receiving surgical sterilization. Based on a 7-year
average life cycle for cats and a 10-year average life cycle for
dogs, approximately 10.53 MM companion animals a year require
sterilization. The current cost of sterilization, and therefore the
potential yearly total revenue as well as market size, is $443.3 MM
for cats and $519.5 MM for dogs, or $962.7 MM total, according to
the AVMA.
[0005] Four to six million healthy companion animals are euthanized
every year according to Humane Society statistics at a cost to
local governments throughout the US of $2 billion. This cost to
local government is increased by populations of larger mammals,
especially deer, which both spread disease as well as cause the
insurance industry millions of dollars yearly due to injury to
person and property.
[0006] Currently, the only method of pet sterilization is surgical.
Single procedures range in cost from $50-$150. There is no
single-injection sterilization product currently on the market.
However, there does exist a drug called Leupron, which causes
temporarily sterilization, and is used on humans to treat prostate
and breast cancer. The drawbacks of Leupron are its cost, about
$2,000 per injection, and the fact that it is only a temporary
solution and thus must be readministered. Hormonal contraceptive
treatments, involving injections, sprays, or lotions all
temporarily remove the ability to reproduce, yet require repeated
dosages. They currently are used primarily in older animals, and
represent a very small fraction of the market.
[0007] It is often difficult to deliver compounds, such as
proteins, peptides, genetic material, and other drugs and
diagnostic compounds intracellularly because cell membranes resist
the passage of these compounds. Various methods have been developed
to administer agents intracellularly. For example, genetic material
has been administered into cells in vivo, in vitro, and ex vivo
using viral vectors, DNA/lipid complexes, and liposomes. While
viral vectors are efficient, questions remain regarding the safety
of a live vector and the development of an immune response
following repeated administration. Lipid complexes and liposomes
appear less effective at transfecting DNA into the nucleus of the
cell and potentially may be destroyed by macrophages in vivo.
[0008] Considerable interest exists with respect to the subject of
sterilization of animals. This is especially true of those
concerned with veterinary medicine and animal husbandry,
particularly as they relate to the subject of sterilization of
domestic animals. Various methods have been developed over the
years to accomplish sterilization. For example, with respect to
male cattle, the most widely used procedure for eliminating
problems of sexual or aggressive behavior is sterilization through
surgical castration. This is done in various ways, e.g., crushing
the spermatic cord, retaining the testes in the inguinal ring, or
use of a rubber band, placed around the neck of the scrotum, to
cause sloughing off of the scrotum and testes. However most of
these "mechanical" castration methods have proven to be undesirable
in one respect or another; for example they (1) are traumatic, (2)
introduce the danger of anesthesia, (3) are apt to produce
infection, and (4) require trained personnel. Moreover, all such
mechanical castration methods result in complete abolition of the
testes and this of course implies complete removal of the anabolic
effects of any steroids which are produced by the testes and which
act as stimuli to growth and protein deposition.
[0009] These drawbacks have caused consideration of various
alternative sterilization techniques such as the use of chemical
sterilization agents. However, the use of chemical sterilization
agents has its own set of advantages and disadvantages. On the
positive side, chemical sterilization eliminates the stress and
danger associated with mechanical castration. Chemical
sterilization also has the added advantage of allowing for
retention of certain anabolic effects resulting from a continued
presence of low levels of circulating testosterone. This is
especially valuable in the case of animals raised for human
consumption since circulating testosterone promotes growth,
efficiency of feed conversion and protein deposition.
Unfortunately, there are several disadvantages associated with
chemical sterilization. For example chemical sterilization is often
temporary rather than permanent; and it also sometimes produces
extremely severe, and even fatal, side effects. Many of these
chemical sterilization methods have been aimed at regulation of
luteinizing hormone produced at various stages of an animal's
sexual development. Most of the chemicals proposed for such
sterilization purposes are hormones or hormone analogs. For example
U.S. Pat. No. 4,444,759 describes a class of peptides analogous to
GnRH (i.e., gonadotropin-releasing hormone, and particularly
luteinizing hormone-releasing hormone) capable of inhibiting
release of gonadotropins by the pituitary gland and thereby
inhibiting release of the steroidal hormones, estradiol,
progesterone and testosterone.
[0010] Another approach has been to use certain chemicals to
produce antibodies in an animal which exhibit cross-reactivity with
the gonadotropins produced by the animal's pituitary gland. It is
generally thought that with such early antigenic stimulation,
formation of antibodies is more continuously stimulated by the
release of endogenous hormones and that early immunization with
such luteinizing hormone deters the maturation of the gonads and
adnexal glands. This, in turn, is thought to inhibit
spermatogenesis at the spermatogonial level. For example, U.S. Pat.
No. 4,691,006 teaches injection of a compound to elicit formation
of antibodies which exhibit cross-reactivity with the gonadotropins
produced by the animal's pituitary. With early antigenic
stimulation of this kind, the formation of such antibodies is more
continuously stimulated by release of endogenous hormones. Early
immunization with such luteinizing hormone also deters the
maturation of the gonads and adnexal glands.
[0011] Similarly, luteinizing hormone has been administered to
animals after they have attained the age of puberty in order to
atrophy their reproductive organs and to cause a decrease in libido
(see generally, Tallau and Laurence, Fertility and Sterility, 22
(2): 113-118 (1971); Pineda, et al. Proc. Soc. Exper. Biol. Med.
125 (3):665-668 (1967), and Quadri, et al. Proc. Soc. Exp. Biol.
Med., 123:809-814 (1966)). Such treatments also impair
spermatogenesis in noncastrated adult male animals by interruption
of the spermatogenic cycle.
[0012] Other chemical sterilization agents have been specifically
designed for use on female animals. For example, it is well known
that certain antigens will produce an antiserum against a requisite
estrogen. This is accomplished by first making an antigen and then
injecting the antigen into an animal for purposes of antiserum
production. The animal is then bled to recover the antiserum. Any
female animal of the same species as the host animal may then be
injected with the antiserum at the proper time prior to ovulation
and the injected antiserum will cause temporary sterilization of
that animal.
[0013] Other methods of chemical sterilization have been based upon
direct chemical attack upon certain cells of the pituitary itself
(as opposed to chemical attacks upon the hormone products of such
cells) with a view toward permanently destroying such cells. Again,
this approach is suggested by the fact that follicle stimulating
hormone (FSH) and luteinizing hormone (LH) (sometimes referred to
as gonadotropins or gonadotropic hormones) are released by the
pituitary gland to regulate functioning of the gonads to produce
testosterone in the testes and progesterone and estrogen in the
ovaries. They also regulate the production and maturation of
gametes.
[0014] Several chemical agents have been proposed for such
purposes. However, it has been found that most chemical agents
which are in fact capable of destroying the gonadotrophs of an
animal's anterior pituitary gland also tend to produce extremely
toxic side effects which can severely weaken, and sometimes kill,
the treated animal. Hence, with respect to the general subject of
chemical sterilization, any chemical capable of producing
sterilization without, or with minimal, toxic side effects would be
of great value in the fields of animal husbandry, veterinary
medicine and wildlife control.
[0015] Myers, et al. Biochem. J., 227 (1):343 (1985) describes a
sterilization procedure employing a GnRH/diphtheria toxin
conjugate. Singh, et al., Int. J. Pharm. 76: R5-R8 describes
controlled release of LHRH-DT from bioerodible hydrogel
microspheres which induces production of antibodies to GnRH which
then serve to inactivate endogenous LHRH in the circulation. U.S.
Pat. No. 6,326,467 to Nett, et al., describes the use of conjugates
of cytotoxic compounds including daunomycin and other toxins to
analogs of gonadotropin-releasing hormones for sterilization of
animals or to treat certain sex hormone related diseases.
[0016] It is therefore an object of the present invention to
provide compositions and methods for delivery of targeted drugs,
especially cytotoxic compounds targeted to cell by specific
hormones, for sterilization and treatment of disorders, especially
of reproductive tissues.
SUMMARY OF THE INVENTION
[0017] A method and compositions for targeted drug delivery have
been developed. The compositions include a targeting molecule, such
as a hormone that specifically binds to a receptor on the surface
of the targeted cells; a drug to be delivered, such as a toxin that
will kill the targeted cells; and a nanoparticle, which contains on
or within the nanoparticle, the drug to be delivered, as well as
has attached thereto, the targeting molecule. In a preferred
embodiment, the ratio of targeting molecule to toxin or other drug
is in the range of 1:10 or more. This higher density of toxin is
critical to achieving the desired treatment in some cases.
[0018] Examples of targeting molecules include hormones, ligands
for specific cell surface receptors, and antibodies. Examples of
cytotoxic drugs include toxins, BCNU, adriamycin, cisplatin, and
other chemotherapeutic agents, radioactive compounds,
radioisotopes, especially yttrium ("Y"), and substances which
elicit the host to attack tumor cells, and radioactive colloids.
Nanoparticles can consist of drug or drug associated with carrier,
such as a controlled or sustained release materials like a
poly(lactide-co-glycolide), a liposome or surfactant.
[0019] The targeted nanoparticles can be administered in
combination with compositions for improving cellular
internalization using a receptor mediated mechanism are
disclosed.
[0020] The compositions are administered by injections in most
cases, although materials combined with the gel will typically be
applied to cell membranes to achieve high rates of transport of the
compound to be delivered across those membranes, relative to when
non-viscous fluids are used with the enhancers or the viscous
fluids are used alone. Compositions can be applied topically
orally, nasally, vaginally, rectally, and ocularly. Enhancer is
administered systemically or, more preferably, locally.
Compositions can be applied by injection via catheter,
intramuscularly, subcutaneously, and intraperitoneally.
Compositions can also be administered to the pulmonary or
respiratory system, most preferably in an aerosol.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIGS. 1A and 1B are graphs of the association of florescence
nanoparticles, with and without LHRH, with cells as determined by
flow cytometry. FIG. 1A illustrates intensity of cell florescence
(log) obtained when incubating cells with nanoparticles not
conjugated to LHRH as described in Example 2. FIG. 1B shows the
increased intensity of cellular florescence achieved following
exposure of cells to LHRH-conjugated florescence nanoparticles.
DETAILED DESCRIPTION OF THE INVENTION
[0022] A method and compositions have been developed in which a
drug is targeted to a specific cell via a receptor on the surface
of the targeted cells. The drug is encapsulated within and/or bound
to a nanoparticle to which the targeting molecule is also bound.
The compositions are then used to deliver medication to a specific
group of cells in the body. Hormones are a preferred targeting
molecule because they travel throughout the systemic circulation
and thus reach all parts of the body; however, they have
physiological effects only on cells which are capable of
`recognizing` their proximity through the presence of biological
`receptors` embedded within the cell membrane. Each hormone has its
own specific type of receptor; insulin, for example, will only
cause a change in biological function in cells that possess
specific insulin receptors.
[0023] In some cases, a biological change in cell function is
effected as soon as the hormone attaches to its receptor on the
membrane; in other cases, physiological effects will only be
observed when the hormone attaches to the receptor and,
additionally, enters the cell. This latter process is known as
`receptor-mediated endocytosis` and is the key to highly selective
hormonal targeting of certain cell types. For example, the method
takes advantage of the fact that in all mammals, cells in the
anterior pituitary gland naturally recognize and absorb a hormone
called luteinizing-hormone-releasing-hormone (LHRH). The anterior
pituitary cells (known as gonadotrope cells) are subsequently
responsible for the release into the bloodstream of hormones that
enable sexual function. The method and compositions essentially
uses LHRH as a `key` to unlock a pathway into these cells. Attached
to the key is a polymer `keychain`, and swinging on the end of that
keychain is a `payload` in the form of a tiny complex called a
nanoparticle. The nanoparticle can be thought of as a tiny
spherical shell; encapsulated within that shell is a toxin that is
to be delivered to the gonadotrope cells. The nanoparticle by
definition will be extremely small in relation to the cell being
entered. It enters the cell along with its toxic payload and then
degrades after a time that can be predetermined when engineering
the nanoparticle in the laboratory. As it degrades, the
nanoparticle releases its payload and the toxin destroys the
gonadotrope cell. The death of the cell means that it can no longer
produce the hormones that enable sexual function. Without the
presence of those hormones in the body, the animal is effectively
sterile, or, in the case of a tumor of a reproductive tissue such
as the testes, prostate, or ovaries, the tumor is killed. Since the
pituitary cells do not regrow with time, this is an irreversible
procedure that is marketable as a humane and surgery-free
alternative to ovariectomies and orchiectomies currently performed
by veterinary surgeons. The gonadotrope cells are the only known
cells within the mammalian body that express receptors for LHRH.
The compound does not cross the blood-brain barrier.
[0024] The nanoparticle both protects the chemical toxin from being
degraded before it reaches the target cell and ensures there is no
`leakage` of the chemical into the bloodstream, thereby protecting
other cells from its harmful effects. Additionally, the tiny
dimensions of the nanoparticle serve to make certain that it will
be carried into the target cell alongside the LHRH without
difficulty. The size of the nanoparticle can be engineered to
enhance uptake of the toxin into the cell.
[0025] Other embodiments are created by substituting another
hormone in order to target other cells, and other kinds of drugs.
One area is in the targeting of human cancerous cells in malignant
tumors. It is known that growing cancer cells show an inordinately
high number of transferrin receptors (on the order of fifteen times
the number found on healthy cells).
[0026] In still another embodiment, delivery is enhanced through
the use of hydrogels of a certain viscosity to enable transport of
drugs across epithelial membranes. If one adds a hydrogel of
viscosity identical to that of the epithelial cell cytoplasm, the
resulting rate of transport of LHRH into the cell and across the
epithelial barrier will be optimized. This opens up the potential
for an application along the lines of an eye gel used to treat
glaucoma. A drug such as epinephrin (a recognized glaucoma
treatment) could be locked inside a nanoparticle and then the
original LHRH key could carry it into the eye and to the active
drug site within the nasolacrimal gland. Topical eye medications
have traditionally been difficult to design because they are washed
out of the eye so quickly; a hydrogel would enhance the rate at
which the drug enters the system and thus markedly reduce the
number of drug applications required to control the disease. At
present, patients are required to use eye drops or creams as often
as once every six hours to ensure the intra-ocular pressure is
maintained at a safe level.
[0027] I. Compositions
[0028] A. Agents to be Delivered
[0029] Therapeutic, diagnostic or prophylactic agents can be
specifically delivered using the technology described herein.
[0030] Any of a variety of therapeutic, diagnostic or prophylactic
agents can be incorporated within the particles, or used to prepare
particles consisting solely of the agent and surfactant. The
particles can be used to locally or systemically deliver a variety
of incorporated agents to a targeted tissue of an animal. Examples
include synthetic inorganic and organic compounds, proteins and
peptides, polysaccharides and other sugars, lipids, and DNA and RNA
nucleic acid sequences having therapeutic, prophylactic or
diagnostic activities. Nucleic acid sequences include genes,
antisense molecules which bind to complementary DNA to inhibit
transcription, and ribozymes. The agents to be incorporated can
have a variety of biological activities, such as vasoactive agents,
neuroactive agents, hormones, anticoagulants, immunomodulating
agents, cytotoxic agents, prophylactic agents, antibiotics,
antivirals, antisense, antigens, and antibodies. In some instances,
the proteins may be antibodies or antigens which otherwise would
have to be administered by injection to elicit an appropriate
response. Compounds with a wide range of molecular weight can be
encapsulated, for example, between 100 and 500,000 grams or more
per mole. As used herein, proteins are defined as consisting of 100
amino acid residues or more; peptides are less than 100 amino acid
residues. Unless otherwise stated, the term protein refers to both
proteins and peptides. Examples include insulin and other hormones.
Polysaccharides, such as heparin, can also be administered.
[0031] Examples of cytotoxic drugs include toxins, BCNU,
adriamycin, cisplatin, and other chemotherapeutic agents,
radioactive compounds, radioisotopes, especially yttrium ("Y"), and
substances which elicit the host to attack tumor cells (Halpern, et
al., J. Nucl. Med. 29:1688-1696 (1988); Quadri, et al., Nucl. Med.
Biol. 20:559-570 (1993); Wang, et al., Radiat. Res. 141:292-302
(1995)), oligonucleotides (Mujoo, et al., Oncogene 12:1617-1623
(1996)), cytokines (Markman, Semin. Oncol. 18:248-254 (1991);
Dedrick, et al., Cancer. Treat Rep. 62:1-11 (1978)), and
radioactive colloids (Rowlinson, et al., Cancer Res. 47:6528-6531
(1987)). Preferred toxins include: diphtheria toxin, ricin toxin,
abrin toxin, pseudomonas exotoxin, shiga toxin, .alpha.-amanitin,
pokeweed antiviral protein (PAP), ribosome inhibiting proteins
(RIP), especially the ribosome inhibiting proteins of barley,
wheat, flax, corn, rye, gelonin, abrin, modeccin and certain
cytotoxic chemicals such as, for example, melphalan, methotrexate,
nitrogen mustard, doxorubicin and daunomycin. These may be
administered as intact molecules or as subunits, as appropriate.
TABLE I below gives some representative "whole" and "modified"
toxins. Some of these toxin types (e.g., bacterial and plant
toxins) also can be further characterized by their possession of
so-called "A-chain" and "B-chain" groups in their molecular
structures. It also should be noted that the toxic domain is often
referred to as the "A-chain" portion of the toxin molecule while
the toxic domain, translocation domain and cell-binding domain are
often collectively referred to as the "whole" toxin or the A-chain
plus the B-chain molecules. For example, such further
classifications could be made according to the attributes,
categories and molecular sizes noted in TABLE I below (wherein the
letters A and B represent the presence of A-chains or B-chains and
the letter K designates the symbol ("kilodalton" used to designate
molecular sizes of such molecules):
1TABLE I Toxins Single Chain Toxins Pokeweed antiviral protein
Gelonin ribosome-inhibiting protein (RIP) Wheat RIP Barley RIP Corn
RIP Rye RIP Flax RIP Bacterial Toxins Diphtheria toxin (whole)
having a toxic domain, a translocation domain and a cell-binding
domain = 62K Diphtheria toxin (modified) having a toxic domain and
a translocation domain = 45K Pseudomonas exotoxin (whole) having a
toxic domain, a translocation domain and a cell-binding domain =
66K Pseudomonas exotoxin (modified) having a toxic domain and a
translocation domain = 40K Shiga toxin (whole) having a toxic
domain, a translocation domain and a cell binding domain = 68K
Shiga toxin (modified) having a toxic domain = 30K Plant Toxins
Ricin A + B (whole) = 62K Ricin A = 30K Abrin A + B = 62K Abrin A =
30K Modeccin A + B = 56K Modeccin A = 26K Small Chemical Toxins
Melphalan Methotrexate Nitrogen Mustard Daunomycin Doxorubicin
[0032] Hormones include peptide-releasing hormones such as insulin,
luteinizing hormone releasing hormone ("LHRH"), gonadotropin
releasing hormone ("GnRH"), deslorelin and leuprolide acetate,
oxytocin, vasoactive intestinal peptide (VIP), glucagon,
parathyroid hormone (PTH), thyroid stimulating hormone, follicle
stimulating hormone, growth factors such as nerve growth factor
(NGF), epidermal growth factor (EGF), vascular endothelial growth
factor (VEGI), insulin-like growth factors (IGF-I and IGF-II),
fibroblast growth factors (FGFs), platelet-derived endothelial cell
growth factor (PD-ECGF), transforming growth factor beta
(TGF-.beta.), and keratinocyte growth factor (KGF). Other materials
which can be delivered include cytokines such as tumor necrosis
factors (TFN-.alpha. and TNF-.beta.), colony stimulating factors
(CSFs), interleukin-2, gamma interferon, consensus interferon,
alpha interferons, beta interferon; attachment peptides such as
RGD; bioactive peptides such as renin inhibitory peptides,
vasopressin, detirelix, somatostatin, and vasoactive intestinal
peptide; coagulation inhibitors such as aprotinin, heparin, and
hirudin; enzymes such as superoxide dismutase, neutral
endopeptidase, catalase, albumin, calcitonin, alpha-1-antitrypsin
(A1A), deoxyribonuclease (DNAase), lectins such as concanavalin A,
and analogues thereof.
[0033] Diagnostic agents can also be delivered. These can be
administered alone or coupled to one or more bioactive compounds as
described above. The agents can be radiolabelled, fluorescently
labeled, enzymatically labeled and/or include magnetic compounds
and other materials that can be detected using x-rays, ultrasound,
magnetic resonance imaging ("MRI"), computed tomography ("CT"), or
fluoroscopy.
[0034] Genes for the treatment of diseases such as cystic fibrosis
can be administered, as can beta agonists for asthma.
[0035] Those therapeutic agents which are charged, such as most of
the proteins, including insulin, can be administered as a complex
between the charged therapeutic agent and a molecule of opposite
charge. Preferably, the molecule of opposite charge is a charged
lipid or an oppositely charged protein.
[0036] Any of a variety of diagnostic agents can be incorporated
within the particles, which can locally or systemically deliver the
incorporated agents following administration to a patient. Any
biocompatible or pharmacologically acceptable gas can be
incorporated into the particles or trapped in the pores of the
particles using technology known to those skilled in the art. The
term gas refers to any compound which is a gas or capable of
forming a gas at the temperature at which imaging is being
performed. In one embodiment, retention of gas in the particles is
improved by forming a gas-impermeable barrier around the particles.
Such barriers are well known to those of skill in the art.
[0037] Other imaging agents which may be utilized include
commercially available agents used in positron emission tomography
(PET), computer assisted tomography (CAT), single photon emission
computerized tomography, x-ray, fluoroscopy, and magnetic resonance
imaging (MRI).
[0038] Examples of suitable materials for use as contrast agents in
MRI include the gadolinium chelates currently available, such as
diethylene triamine pentacetic acid (DTPA) and gadopentotate
dimeglumine, as well as iron, magnesium, manganese, copper and
chromium.
[0039] Examples of materials useful for CAT and x-rays include
iodine based materials for intravenous administration, such as
ionic monomers typified by diatrizoate and iothalamate, non-ionic
monomers such as iopamidol, isohexol, and ioversol, non-ionic
dimers, such as iotrol and iodixanol, and ionic dimers, for
example, ioxagalte.
[0040] Excipients
[0041] In addition to a therapeutic or diagnostic agent (or
possibly other desired molecules for delivery), the particles can
include, and preferably, do include, one or more of the following
excipients; a sugar, such as lactose, a protein, such as albumin,
and/or a surfactant.
[0042] B. Carriers
[0043] The drug and targeting molecule are linked to a common
carrier, preferably a nanoparticle, which must be able to bind to a
cell surface receptor. As used herein, a "nanoparticle" includes
liposomes, micelles, drug particles and polymeric particles, having
a dimension of less than one micron, more preferably in the range
of less than or equal to 200 nm, although nanoparticles may be in
the range of as large as approximately 500 nm, and as small as a
few nm. Liposomes consist basically of a phospholipid bilayer
forming a shell around an aqueous core. Advantages include the
lipophilicity of the outer layers which "mimic" the outer membrane
layers of cells and that they are taken up relatively easily by a
variety of cells. Polymeric vehicles typically consist of
nanospheres and nanocapsules formed of biocompatible polymers,
which are either biodegradable (for example, polylactic acid) or
non-biodegradable (for example, ethylenevinyl acetate). Some of the
advantages of the polymeric devices are ease of manufacture and
high loading capacity, range of size from nanometer to micron
diameter, as well as controlled release and degradation profile.
Both liposomes and small polymeric vehicles are referred to herein
as "nanoparticles", unless specifically stated otherwise.
[0044] The particles can be prepared entirely from a therapeutic or
diagnostic agent, or from a combination of the agent and a
surfactant, excipient or polymeric material. The particles
preferably are biodegradable and biocompatible, and optionally are
capable of biodegrading at a controlled rate for delivery of a
therapeutic or diagnostic agent. The particles can be made of a
variety of materials. Both inorganic and organic materials can be
used. Polymeric and non-polymeric materials, such as fatty acids,
may be used. Other suitable materials include, but are not limited
to, gelatin, polyethylene glycol, trehalose, and dextran. Particles
with degradation and release times ranging from seconds to months
can be designed and fabricated, based on factors such as the
particle material.
[0045] Polymeric Particles
[0046] Polymeric particles may be formed from any biocompatible,
and preferably biodegradable polymer, copolymer, or blend. The
polymers may be tailored to optimize different characteristics of
the particle including: i) interactions between the agent to be
delivered and the polymer to provide stabilization of the agent and
retention of activity upon delivery; ii) rate of polymer
degradation and, thereby, rate of drug release profiles; iii)
surface characteristics and targeting capabilities via chemical
modification; and iv) particle porosity.
[0047] Surface eroding polymers such as polyanhydrides may be used
to form the particles. For example, polyanhydrides such as
poly[(p-carboxyphenoxy)-hexane anhydride] (PCPH) may be used.
Biodegradable polyanhydrides are described in U.S. Pat. No.
4,857,311.
[0048] In another embodiment, bulk eroding polymers such as those
based on polyesters including poly(hydroxy acids) can be used. For
example, polyglycolic acid (PGA), polylactic acid (PLA), or
copolymers thereof may be used to form the particles. The polyester
may also have a charged or functionalizable group, such as an amino
acid. In a preferred embodiment, particles with controlled release
properties can be formed of poly(D,L-lactic acid) and/or
poly(D,L-lactic-co-glycolic acid) ("PLGA") which incorporate a
surfactant such as DPPC.
[0049] Other polymers include polyamides, polycarbonates,
polyalkylenes such as polyethylene, polypropylene, poly(ethylene
glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly
vinyl compounds such as polyvinyl alcohols, polyvinyl ethers, and
polyvinyl esters, polymers of acrylic and methacrylic acids,
celluloses and other polysaccharides, and peptides or proteins, or
copolymers or blends thereof. Polymers may be selected with or
modified to have the appropriate stability and degradation rates in
vivo for different controlled drug delivery applications.
[0050] In one embodiment, particles are formed from functionalized
polyester graft copolymers, as described in Hrkach et al.,
Macromolecules, 28:4736-4739 (1995); and Hrkach et al.,
"Poly(L-Lactic acid-co-amino acid) Graft Copolymers: A Class of
Functional Biodegradable Biomaterials" in Hydrogels and
Biodegradable Polymers for Bioapplications, ACS Symposium Series
No. 627, Raphael M. Ottenbrite et al., Eds., American Chemical
Society, Chapter 8, pp. 93-101, 1996.
[0051] Materials other than biodegradable polymers may be used to
form the particles. Suitable materials include various
non-biodegradable polymers and various excipients. The particles
also may be formed of a therapeutic or diagnostic agent and
surfactant alone. In one embodiment, the particles may be formed of
the surfactant and include a therapeutic or diagnostic agent.
[0052] Polymeric particles may be prepared using single and double
emulsion solvent evaporation, spray drying, solvent extraction,
solvent evaporation, phase separation, simple and complex
coacervation, interfacial polymerization, and other methods well
known to those of ordinary skill in the art. Particles may be made
using methods for making microspheres or microcapsules known in the
art, provided that the conditions are optimized for forming
particles with the desired diameter.
[0053] Methods developed for making microspheres for delivery of
encapsulated agents are described in the literature, for example,
as described in Doubrow, M., Ed., "Microcapsules and Nanoparticles
in Medicine and Pharmacy," CRC Press, Boca Raton, 1992. Methods
also are described in Mathiowitz and Langer, J. Controlled Release
5,13-22 (1987); Mathiowitz et al., Reactive Polymers 6, 275-283
(1987); and Mathiowitz et al., J. Appl. Polymer Sci. 35, 755-774
(1988). The selection of the method depends on the polymer
selection, the size, external morphology, and crystallinity that is
desired, as described, for example, by Mathiowitz et al., Scanning
Microscopy 4: 329-340 (1990); Mathiowitz et al., J. Appl. Polymer
Sci. 45, 125-134 (1992); and Benita et al., J. Pharm. Sci. 73,
1721-1724 (1984).
[0054] In solvent evaporation, described for example, in Mathiowitz
et al., (1990), Benita; and U.S. Pat. No. 4,272,398 to Jaffe, the
polymer is dissolved in a volatile organic solvent, such as
methylene chloride. Several different polymer concentrations can be
used, for example, between 0.05 and 1.0 g/ml. The therapeutic or
diagnostic agent, either in soluble form or dispersed as fine
particles, is added to the polymer solution, and the mixture is
suspended in an aqueous phase that contains a surface active agent
such as poly(vinyl alcohol). The aqueous phase may be, for example,
a concentration of 1% poly(vinyl alcohol) w/v in distilled water.
The resulting emulsion is stirred until most of the organic solvent
evaporates, leaving solid microspheres, which may be washed with
water and dried overnight in a lyophilizer. Microspheres with
different sizes (between 1 and 1000 microns) and morphologies can
be obtained by this method.
[0055] Solvent removal was primarily designed for use with less
stable polymers, such as the polyanhydrides. In this method, the
agent is dispersed or dissolved in a solution of a selected polymer
in a volatile organic solvent like methylene chloride. The mixture
is then suspended in oil, such as silicon oil, by stirring, to form
an emulsion. Within 24 hours, the solvent diffuses into the oil
phase and the emulsion droplets harden into solid polymer
microspheres. Unlike the hot-melt microencapsulation method
described for example in Mathiowitz et al., Reactive Polymers,
6:275 (1987), this method can be used to make microspheres from
polymers with high melting points and a wide range of molecular
weights. Microspheres having a diameter for example between one and
300 microns can be obtained with this procedure.
[0056] With some polymeric systems, polymeric particles prepared
using a single or double emulsion technique vary in size depending
on the size of the droplets. If droplets in water-in-oil emulsions
are not of a suitably small size to form particles with the desired
size range, smaller droplets can be prepard, for example, by
sonication or homogenation of the emulsion, or by the addition of
surfactants.
[0057] If the particles prepared by any of the above methods have a
size range outside of the desired range, particles can be sized,
for example, using a sieve, and further separated according to
density using techniques known to those of skill in the art.
[0058] The polymeric particles can be prepared by spray drying.
Methods of spray drying, such as that disclosed in PCT WO 96/09814
by Sutton and Johnson, disclose the preparation of smooth,
spherical microparticles of a water-soluble material with at least
90% of the particles possessing a mean size between 1 and 10
.mu.m.
[0059] Liposomes
[0060] Liposomes can be produced by standard methods such as those
reported by Kim, et al., Biochim. Biophys. Acta 728, 339-348
(1983); Liu, D., et al., Biochim. Biophys. Acta 1104, 95-101
(1992); and Lee, et al., Biochim. Biophys. Acta., 1103, 185-197
(1992)). Many liposome formulations using many different lipid
components have been used in various in vitro cell culture and
animal experiments. Parameters have been identified that determine
liposomal properties and are reported in the literature, for
example, by Lee, K. D., et al. Biochim. Biophys. Acta., 1103,
185-197 (1992); Liu, D., Mori, A. and Huang, L., Biochim. Biophys.
Acta, 1104, 95-101 (1992); Wang, C. Y. and Huang, L., Biochem., 28,
9508-9514 (1989)).
[0061] Briefly, the lipids of choice, dissolved in an organic
solvent, are mixed and dried onto the bottom of a glass tube under
vacuum. The lipid film is rehydrated using an aqueous buffered
solution containing the material to be encapsulated by gentle
swirling. The hydrated lipid vesicles or liposomes are washed by
centrifugation and can be filtered and stored at 4.degree. C. This
method is described in more detail in Thierry, A. R. and
Dritschilo, A "Intracellular availability of unmodified,
phosphorothioated and liposomally encapsulated
oligodeoxynucleotides for antisense activity" Nuc. Ac. Res.
20:5691-5698 (1992).
[0062] A nanoparticle system carrying a toxin payload can be made
using the procedure as described in: Pautot, Frisken, Weitz
"Production of unilamellar vesicles using an inverted emulsion"
(submitted for publication). Using Pautot et al's technique,
streptavidin-coated lipids (DPPC, DSPC, and similar lipids) can be
used to manufacture liposomes. The drug encapsulation technique
described by Needham D, Newhirst M W. Advanced Drug Delivery
Reviews, 53 (3): 285-305 Dec. 31 2001), can be used to load these
vesicles with doxorubicin, a model small-molecule toxin currently
used in cancer therapy.
[0063] The liposomes can be prepared by exposing chloroformic
solution of various lipid mixtures to high vacuum and subsequently
hydrating the resulting lipid films (DSPC/CHOL) with pH 4 buffers,
and extruding them through polycarbonated filters, after a freezing
and thawing procedure. It is possible to use DPPC supplemented with
DSPC or cholesterol to increase encapsulation efficiency or
increase stability, etc. A transmembrane pH gradient is created by
adjusting the pH of the extravesicular medium to 7.5 by addition of
an alkalinization agent. The anticancer drug doxorubicin is
subsequently entrapped by addition of the drug solution in small
aliquots to the vesicle solution, at an elevated temperature, to
allow drug accumulation inside the liposomes.
[0064] Complex Forming Materials
[0065] If the agent to be delivered is negatively charged (such as
insulin), protamine or other positively charged molecules can be
added to provide a lipophilic complex which results in the
sustained release of the negatively charged agent. Negatively
charged molecules can be used to render insoluble positively
charged agents.
[0066] Materials Enhancing Sustained Release
[0067] If the molecules are hydrophilic and tend to solubilize
readily in an aqueous environment, another method for achieving
sustained release is to use cholesterol or very high surfactant
concentration.
[0068] Surfactants
[0069] Surfactants which can be incorporated into particles include
phosphoglycerides. Exemplary phosphoglycerides include
phosphatidylcholines, such as the naturally occurring surfactant,
L-.alpha.-phosphatidylcholine dipalmitoyl ("DPPC"). The surfactants
advantageously improve surface properties by, for example, reducing
particle-particle interactions, and can render the surface of the
particles less adhesive. The use of surfactants endogenous to the
lung may avoid the need for the use of non-physiologic
surfactants.
[0070] As used herein, the term "surfactant" refers to any agent
which preferentially absorbs to an interface between two immiscible
phases, such as the interface between water and an organic polymer
solution, a water/air interface or organic solvent/air interface.
Surfactants generally possess a hydrophilic moiety and a lipophilic
moiety, such that, upon absorbing to microparticles, they tend to
present moieties to the external environment that do not attract
similarly-coated particles, thus reducing particle agglomeration.
Surfactants may also promote absorption of a therapeutic or
diagnostic agent and increase bioavailability of the agent.
[0071] As used herein, a particle "incorporating a surfactant"
refers to a particle with a surfactant on at least the surface of
the particle. The surfactant may be incorporated throughout the
particle and on the surface during particle formation, or may be
coated on the particle after particle formation. The surfactant can
be coated on the particle surface by adsorption, ionic or covalent
attachment, or physically "entrapped" by the surrounding matrix.
The surfactant can be, for example, incorporated into controlled
release particles, such as polymeric microspheres.
[0072] Providing a surfactant on the surfaces of the particles can
reduce the tendency of the particles to agglomerate due to
interactions such as electrostatic interactions, Van der Waals
forces, and capillary action. The presence of the surfactant on the
particle surface can provide increased surface rugosity
(roughness), thereby improving aerosolization by reducing the
surface area available for intimate particle-particle interaction.
The use of a surfactant which is a natural material of the lung can
potentially reduce opsonization (and thereby reducing phagocytosis
by alveolar macrophages), thus providing a longer-lived controlled
release particle in the lung.
[0073] Surfactants known in the art can be used including any
naturally occurring surfactant. Other exemplary surfactants include
diphosphatidyl glycerol (DPPG); hexadecanol; fatty alcohols such as
polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a
surface active fatty acid, such as palmitic acid or oleic acid;
sorbitan trioleate (Span 85); glycocholate; surfactin; a poloxomer;
a sorbitan fatty acid ester such as sorbitan trioleate; tyloxapol
and a phospholipid.
[0074] C. Targeting of Particles
[0075] The nanoparticles are targeted so that they are delivered to
a particular cell type. It is well known in the art how to modify
carriers such that they are bound, ionically or covalently, to a
ligand (i.e., LHRH) that binds to a cell surface receptor. Examples
of materials used to covalently bind targeting molecules to the
materials forming the carrier including 2-iminothiolane,
N-succinimidyl-3-(2-pyridyldithio)prop- rionate (SPDP),
4-succinimidyloxycarbonyl-.alpha.-(2-pyridyldithio)-toluen- e
(SMPT), m-maleimidobenzoyl-N-hydroxysuccinimide-ester (MBS),
N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl
4-(p-maleimidophenyl)butyrate (SMPB),
1-ethyl-3-(3-dimethylaminopropyl)ca- rbodiimide (EDC),
bis-diazobenzidine and glutaraldehyde. Other means of coupling
targeting molecules can be used with materials such as liposomes.
For example, U.S. Pat. No. 5,258,499 to Konigsberg et al. describes
the incorporation of receptor specific ligands into liposomes,
which are then used to target receptors on the cell surface. The
use of carriers can be particularly important for intracellularly
delivering nucleic acid molecules. In one embodiment, nucleic acid
molecules are encapsulated in a liposome, preferably a cationic
liposome, that has a receptor-binding ligand, such as LHRH, on its
surface. The liposome is then dispersed in a viscous fluid. When
the composition is administered, the liposomes are endocytosed by
the cell, and the nucleic acid molecules are released from the
liposome inside the cell.
[0076] Examples of targeting molecules include hormones, ligands
for specific cell surface receptors, and antibodies. A key criteria
is that the targeting molecule specifically binds to a receptor on
the surface of the cells where the drug is to be delivered.
Moreover, the targeting molecule should preferably assist in uptake
of the drug to be delivered into the cell, as in the case of
hormone binding to receptors which result in endocytosis of the
nanoparticle containing the drug into the cell.
[0077] Targeting molecules can be attached to the particles via
reactive functional groups on the particles. For example, targeting
molecules can be attached to the amino acid groups of
functionalized polyester graft copolymer particles, such as
poly(lactic acid-co-lysine) (PLAL-Lys) particles. Targeting
molecules permit binding interaction of the particle with specific
receptor sites, such as those within the lungs. The particles can
be targeted by attachment of ligands which specifically or
non-specifically bind to particular targets. Exemplary targeting
molecules include antibodies and fragments thereof including the
variable regions, lectins, and hormones or other organic molecules
capable of specific binding, for example, to receptors on the
surfaces of the target cells.
[0078] The binding of ligands or assembly proteins to surface
receptors of eucaryotic cell membranes has been extensively studied
in an effort to develop better ways to promote or enhance cellular
uptake. For example, binding of ligands or proteins has been
reported to initiate or accompany a cascade of nonequilibrium
phenomena culminating in the cellular invagination of membrane
complexes within clathrin-coated vesicles (Goldstein, et al., Ann.
Rev. Cell Biol. 1:1-39 (1985); Rodman, et al., Curr. Op. Cell Biol.
2:664-72 (1990); Trowbridge, Curr. Op. Cell Biol. 3:634-41 (1991);
Smythe, et al., J. Cell Biol. 108:843-53 (1989); Smythe, et al., J.
Cell Biol. 119:1163-71 (1992); and Schmid, Curr. Op. Cell Biol.
5:621-27 (1993)). This process has been referred to as
receptor-mediated endocytosis ("RME"). Beyond playing a central
role in cellular lipid trafficking (Pagano, Curr. Op. Cell Biol.
2:652-63 (1990)), RME is the primary means by which macromolecules
enter eucaryotic cells. An effective strategy for enhancing the
uptake of cytotoxic and therapeutic drugs involves exploiting the
rapidity and specificity of transmembrane transport via
receptor-mediated endocytosis (Goldstein, et al., Ann. Rev. Cell
Biol. 1:1-39 (1985)) by targeting receptors on the plasma membranes
of endothelial (Barzu, et al., Biochem. J. 15; 238 (3):847-854
(1986); Magnusson & Berg, Biochem. J. 257:65-56 (1989)),
phagocytic (Wright & Detmers, "Receptor-mediated phagocytosis"
in The Lung: Scientific Foundations (Crystal, et al., eds.), pp.
539-49 (Ravens Press, Ltd., New York, N.Y. (1991)); and tumor
cells, as well as cells of other tissues.
[0079] D. Gels for Enhancement of Uptake into Cells.
[0080] It has been demonstrated that, by embedding individual cell
populations in hydrogel media of macroscopic viscosity similar to
that characteristic of cell cytoskeleta, the rate of
receptor-mediated endocytosis can be significantly enhanced
(Edwards, et al., Proc. Natl. Acad. Sci. U.S.A. 93:1786-91 (1996);
PCT US97/03276 by Massachusetts Institute of Technology and
Pennsylvania State University Foundation). This enhancement effect
appears to reflect a fluid-mechanical origin of receptor-mediated
endocytosis, involving the rapid expansion of plasma membrane in
the vicinity of a receptor cluster leading to an invaginating
membrane motion that is sensitive to the viscous properties of the
extracellular environment (Edwards, et al., Proc. Natl. Acad. Sci.
U.S.A. 93:1786-91 (1996); Edwards, et al., Biophys. J. 71:1208-14
(1996)).
[0081] When the differences between the apparent viscosities of the
cytosolic fluid and the extracellular fluid are extremely large,
membrane deformation is strongly resisted and the initial thrust of
the membrane is damped. However, as the differences between the
apparent viscosities of the cytosolic fluid and the extracellular
fluid become extremely small, membrane deformation becomes
progressively rapid. Accordingly, the rate of endocytosis can be
increased by adjusting the viscosity of the extracellular fluid so
that it is approximately the same as that of the cytosolic fluid,
as described by PCT/US97/03276. If the viscosity of the
extracellular fluid is appreciably higher or lower than that of the
cytosolic fluid, the rate of endocytosis decreases.
[0082] Clustering of membrane complexes is favorable for rapid
internalization. The rate of internalization can be increased in
proportion to the magnitude of binding energy. This is due, in
part, to the specificity of receptors to particular ligands and/or
adaptor proteins. Clustering of complexes occurs in the vicinity of
pits to which clathrin triskelions absorb from the cytosolic side
of the cell membrane and subsequently polymerize to form a clathrin
coat. Some clustering has also been observed in the vicinity of
caveolae, or non-clathrin-coated pits. The membrane-tension
depression occurring within the vicinity of an evolving pit,
originating in the process of membrane complexation, is directly
proportional to the number of membrane complexes formed within that
pit. In general, clustered complexes have been found to internalize
substances more rapidly than nonclustered complexes.
[0083] The magnitudes of apparent viscosity difference and receptor
clustering have each been found to alter the rate of RME. Membrane
tension can also be manipulated to influence the rate of RME.
Increasing the membrane tension `hardens` the cell membrane, making
cell membrane depression increasingly prohibitive. This phenomenon
has been commented upon by Sheetz, M. P. and Dai, J. (1995),
presented at the 60th Annual Cold Spring Harbor Symposium on
Protein Kinases, Cold Spring Harbor, N.Y., on the basis of studies
that show an increased rate of endocytosis for neuronal growth
cones coinciding with membrane tension lowering.
[0084] Accordingly, the rate of internalization can be increased by
a) adjusting the viscosity of the extracellular fluid to
approximate that of the cytosolic fluid; b) forming complexes of
the material to be internalized; and c) reducing membrane tension.
Compositions and methods for increasing the rate of endocytosis are
described in detail below.
[0085] Suitable viscous fluids for use in intracellularly
administering compounds include biocompatible hydrogels, lipogels,
and highly viscous sols. A hydrogel is defined as a substance
formed when an organic polymer (natural or synthetic) is
cross-linked via covalent, ionic, or hydrogen bonds to create a
three-dimensional open-lattice structure which entraps water
molecules to form a gel. Examples of materials which can be used to
form a hydrogel include polysaccharides, proteins and synthetic
polymers. Examples of polysaccharides include celluloses such as
methyl cellulose, dextrans, and alginate. Examples of proteins
include gelatin and hyaluronic acid. Examples of synthetic polymers
include both biodegradeable and non-degradeable polymers (although
biodegradeable polymers are preferred), such as polyvinyl alcohol,
polyacrylamide, polyphosphazines, polyacrylates, polyethylene
oxide, and polyalkylene oxide block copolymers ("POLOXAMERS.TM.")
such as PLURONICS.TM. or TETRONICS.TM. (polyethylene
oxide-polypropylene glycol block copolymers).
[0086] In general, these polymers are at least partially soluble in
aqueous solutions, such as water, buffered salt solutions, or
aqueous alcohol solutions. Several of these have charged side
groups, or a monovalent ionic salt thereof. Examples of polymers
with acidic side groups that can be reacted with cations are
polyphosphazenes, polyacrylic acids, poly(meth)acrylic acids,
polyvinyl acetate, and sulfonated polymers, such as sulfonated
polystyrene. Copolymers having acidic side groups formed by
reaction of acrylic or methacrylic acid and vinyl ether monomers or
polymers can also be used. Examples of acidic groups are carboxylic
acid groups, sulfonic acid groups, halogenated (preferably
fluorinated) alcohol groups, phenolic OH groups, and acidic OH
groups.
[0087] Examples of polymers with basic side groups that can be
reacted with anions are polyvinyl amines, polyvinyl pyridine,
polyvinyl imidazole, polyvinylpyrrolidone and some imino
substituted polyphosphazenes. The ammonium or quaternary salt of
the polymers can also be formed from the backbone nitrogens or
pendant imino groups. Examples of basic side groups are amino and
imino groups.
[0088] Alginate can be ionically cross-linked with divalent
cations, in water, at room temperature, to form a hydrogel matrix.
An aqueous solution containing the compound to be delivered can be
suspended in a solution of a water soluble polymer, and the
suspension can be formed into droplets which are configured into
discrete microcapsules by contact with multivalent cations.
Optionally, the surface of the microcapsules can be crosslinked
with polyamino acids to form a semipermeable membrane around the
encapsulated materials.
[0089] The polyphosphazenes suitable for cross-linking have a
majority of side chain groups which are acidic and capable of
forming salt bridges with di- or trivalent cations. Examples of
preferred acidic side groups are carboxylic acid groups and
sulfonic acid groups. Hydrolytically stable polyphosphazenes are
formed of monomers having carboxylic acid side groups that are
crosslinked by divalent or trivalent cations such as Ca.sup.2+ or
Al.sup.3+. Polymers can be synthesized that degrade by hydrolysis
by incorporating monomers having imidazole, amino acid ester, or
glycerol side groups. For example, a polyanionic
poly[bis(carboxylatophenoxy)]phosphazene (PCPP) can be synthesized,
which is cross-linked with dissolved multivalent cations in aqueous
media at room temperature or below to form hydrogel matrices.
[0090] Methods for the synthesis of the polymers described above
are known to those skilled in the art. See, for example Concise
Encyclopedia of Polymer Science and Polymeric Amines and Ammonium
Salts, (Goethals, ed.) (Pergamen Press, Elmsford, N.Y. 1980). Many
of these polymers are commercially available.
[0091] Preferred hydrogels include aqueous-filled polymer networks
composed of celluloses such as methyl cellulose, dextrans, agarose,
polyvinyl alcohol, hyaluronic acid, polyacrylamide, polyethylene
oxide and polyoxyalkylene polymers ("poloxamers"), especially
polyethylene oxide-polypropylene glycol block copolymers, as
described in U.S. Pat. No. 4,810,503. Several poloxamers are
commercially available from BASF and from Wyandotte Chemical
Corporation as "Pluronics". They are available in average molecular
weights of from about 1100 to about 15,500.
[0092] As used herein, lipogels are gels with nonaqueous fluid
interstices. Examples of lipogels include natural and synthetic
lecithins in organic solvents to which a small amount of water is
added. The organic solvents include linear and cyclic hydrocarbons,
esters of fatty acids and certain amines (Scartazzini et al. Phys.
Chem. 92:829-33 (1988)). As defined herein, a sol is a colloidal
solution consisting of a liquid dispersion medium and a colloidal
substance which is distributed throughout the dispersion medium. A
highly viscous sol is a sol with a viscosity between approximately
0.1 and 2000 Poise. Other useful viscous fluids include gelatin and
concentrated sugar (such as sorbitol) solutions with a viscosity
between approximately 0.1 and 2000 Poise.
[0093] The apparent viscosity of the extracellular fluid (the
composition) must be approximately equal to the viscosity of the
cytosolic fluid in the cell to which the compounds are to be
administered. One of skill in the art can readily determine or
reasonably estimate of the viscosity of the cytosolic fluid using a
viscometer and measuring the applied stress divided by measured
strain rate at the applied stress that corresponds to the stress
the cell membrane imparts upon the cytosolic and extracellular
fluids during endocytosis. Methods for measuring the cytosolic
viscosity include micropipette methods (Evans & Young, Biophys.
J., 56:151-160 (1989)) and methods involving the motion of
membrane-linked colloids (Wang et al., Science, 260:1124-26 (1993).
Typical cytosol viscosities, measured by these techniques, range
from approximately 50-200 Poise. Once this value is measured, the
viscosity of the composition can be adjusted to be roughly equal to
that viscosity, particularly when measured via routine methods at
the applied stress that corresponds to the stress the cell membrane
imparts upon the cytosolic and extracellular fluids during
endocytosis.
[0094] The viscosity can be controlled via any suitable method
known to those of skill in the art. The method for obtaining a
viscous composition with the desired apparent viscosity is not
particularly limited since it is the value of the apparent
viscosity relative to the target cells which is critical. The
apparent viscosity can be controlled by adjusting the solvent
(i.e., water) content, types of materials, ionic strength, pH,
temperature, polymer or polysaccharide chemistry performed on the
materials, and/or external electric, ultrasound, or magnetic
fields, among other parameters.
[0095] The apparent viscosity of the compositions is controlled
such that it lies in the range of between 0.1 and 2000 Poise,
preferably between 7 and 1000 Poise, and most preferably between 2
and 200 Poise. The apparent viscosity can be measured by a standard
rheometer using an applied stress range of between 1 and 1000
Pascals, preferably between 1 and 500 Pascals, and most preferably
between 1 and 100 Pascals. Further, the viscosity of the
compositions is controlled so that the quotient of (apparent
viscosity of the cytosol of the target cells--apparent viscosity of
the composition) and the apparent viscosity of the cytosol of the
target cells is between approximately -0.1 and 0.3, preferably
between approximately 0 and 0.3, more preferably between
approximately 0 and 0.1, and most preferably between approximately
0 and 0.05.
[0096] The composition can be administered as an only slightly
viscous formulation that becomes more viscous in response to a
condition in the body, such as body temperature or a physiological
stimulus, like calcium ions or pH, or in response to an externally
applied condition, such as ultrasound or electric or magnetic
fields. An example is a temperature sensitive poloxamer which
increases in viscosity at body temperature.
[0097] The following are examples of suitable concentration ranges:
Methocel solutions in the range of between 1.0 and 2.0% (w/w),
polyvinyl alcohol solutions between 5 and 15%, pluronic acid
solutions between 15 and 20% and trehalose solutions between 1 and
5%.
[0098] E. Enhancers
[0099] Compounds that can be attached, covalently or noncovalently,
to the carrier or nanoparticle that either stimulates
receptor-mediated endocytosis (RME) or pinocytosis by binding to
receptors on the plasma membrane, binds specifically to receptors
that undergo RME or pinocytosis independently of this binding, or
at least can be associated chemically or physically with other
molecules or "carriers" that themselves undergo RME or pinocytosis,
are referred to as enhancers for intracellular delivery. Examples
include steroids such as estradiol and progesterone, and some
glucocorticoids. Glucocorticocoids such as dexamethasone,
cortisone, hydrocortisone, prednisone, and others are routinely
administered orally or by injection. Other glucocorticoids include
beclomethasone, dipropianate, betamethasone, flunisolide, methyl
prednisone, para methasone, prednisolone, triamcinolome,
alclometasone, amcinonide, clobetasol, fludrocortisone, diflurosone
diacetate, fluocinolone acetonide, fluoromethalone,
flurandrenolide, halcinonide, medrysone, and mometasone, and
pharmaceutically acceptable salts and mixtures thereof. Other
compounds also bind specifically to receptors on cell surfaces.
Many hormone specific receptors are known. These can all be used to
enhance uptake. Selection of molecules binding to receptors which
are predominantly found on a particular cell type or which are
specific to a particular cell type can be used to impart
selectivity of uptake.
[0100] The enhancer is preferably administered at a time and in an
amount effective to maximize expression of receptors, and
consequently receptor mediated internalization of the compound. The
enhancer can itself be the compound to be delivered. The enhancer
can be administered as part of the formulation containing the
compound to be delivered or prior to or as part of a different
formulation. The enhancer may be administered systemically,
followed by administration of the compound to be delivered directly
to the site where uptake is to occur.
[0101] F. Compositions for Lowering or Raising Membrane Tension
[0102] The efficiency of the method can be increased by lowering
the membrane tension. Suitable methods for lowering membrane
tension include including a biocompatible surface active agent in
the hydrogel, performing exothermic reactions on the cell surface
(i.e., complex formation), and applying an external field to the
cell surface. Suitable biocompatible surface active agents include
surfactin, trehalose, fatty acids such as palmitin and oleic acid,
polyethylene glycol, hexadecanol, and phospholipids such as
phosphatidylcholines and phosphatidylglycerols. Suitable
complex-forming chemical reactions include the reaction of
receptor-binding ligands with cell surface receptors for these
ligands, exothermic reactions such as occur between sodium
salicylate and salicylic acid, and neutralization reactions as
between hydrochloric acid and ammonia (Edwards et al. 1996 Biophys.
J. 71, 1208-1214). External fields that can be applied to a cell
surface to reduce membrane tension include ultrasound, electric
fields, and focused light beams, such as laser beams.
[0103] The rate of cellular internalization can also be increased
by causing the clustering of receptors on the cell membrane. This
can be accomplished, for example, by creating zones on the membrane
where the membrane tension is relatively high, causing the membrane
fluid to flow toward the zone of high membrane tension. This flow
can carry receptors localized in the membrane toward each other,
causing them to cluster.
[0104] II. Methods of Administration
[0105] In the simplest embodiment, the nanoparticle is administered
by injection, into the blood stream, peritoneally, subcutaneously,
or administered by inhalation, intranasal, intravaginal or
topically. The compositions can be applied topically to the vagina,
rectum, nose, eye, ear, mouth and the respiratory or pulmonary
system. Preferably, the compositions are applied directly to the
cells to which the compound is to be delivered, usually in a
topical formulation. Examples of methods of administration include
oral administration, as in a liquid formulation or within solid
foods, topical administration to the skin or the surface of the
eye, intravaginal administration, rectal administration, intranasal
administration, and administration via inhalation. When the
composition is administered orally or by inhalation, it is
preferred that it is administered as a dry powder that hydrates
into a hydrogel of an appropriate viscosity after delivery to the
desired location. After inhalation, for example, the hydrogel
absorbs water to obtain the desired viscosity and then delivers
agents to the respiratory system. When administered orally, a
hydrogel can be selected that does not absorb water under
conditions present in the upper gastrointestinal tract, but which
does absorb water under conditions present in the lower
gastrointestinal tract (i.e., at a pH greater than about 6.5). Such
hydrogels are well known to those of skill in the art. The use of
such compositions can optimize the delivery of agents to the lower
gastrointestinal tract.
[0106] In another embodiment, the nanoparticles are formulated. For
example, in a gel, alone or with enhancer, for simultaneous
administration. Alternatively, as parts of a kit, for separate
administration. The enhancer can be administered simultaneously
with or after administration of the composition including the
viscous gel and agent to be delivered. The administration schedule
(e.g., the interval of time between administering the enhancer and
administering the gel composition) can be readily selected by one
of skill in the art to maximize receptor expression and/or binding
before exposure of the cell surface to the agent to be
delivered.
[0107] The dosage will be expected to vary depending on several
factors, including the patient, the particular bioactive compound
to be delivered, and the nature of the condition to be treated,
among other factors. One of skill in the art can readily determine
an effective amount of the bioactive compound or compounds to
administer to a patient in need thereof
[0108] III. Applications for the Compositions and Methods
[0109] A. Sterilization or killing of reproductive tissue
[0110] The compositions described herein can be used to sterilize
mammals (animals and humans) and/or treat certain sex hormone
related diseases such as cancer of the prostate or cancer of the
breast. The preferred targeting molecules for this application are
various peptide hormone molecules such as certain analogs of
gonadotropin-releasing hormone, GnRH. The preferred agent to be
delivered are the cytotoxic agents. The term gonadotropin-releasing
hormone will usually be abbreviated as "GnRH " and as used herein
include analogs thereof.
[0111] The carriers are specifically targeted to the
gonadotropin-secreting cells of the anterior pituitary gland.
Indeed they are the only cells to which the gonadotropin-releasing
hormone portion of applicant's conjugates will bind. The toxic
compounds serve to permanently destroy a subpopulation of the
anterior pituitary cells and thereby eliminate the gland's ability
to secrete gonadotropins. This direct chemical attack upon the
pituitary gland, in turn, causes the animal's gonads to atrophy and
lose their ability to function for reproductive purposes. Without
functioning gonadotrophs, an animal is not able to secrete
luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and
thus is rendered sterile. Consequently, these compositions have
great potential utility in human medicine as well as in veterinary
medicine. This follows from the fact that there are several
important biological reasons for employing castration and
antifertility drugs in humans. For example, breast and prostate
cancers are but two examples of sex steroid-dependent tumors which
respond to such hormonal manipulation. At present, the only
reliable way to inhibit steroid-dependent tumor growth is through
administration of counter-regulatory hormones (e.g., DES in
prostate cancer), sex-steroid hormone binding inhibitors (e.g.,
tamoxifen in breast cancer) or surgical castration. Thus the
potential medical uses of such chemical castration compounds are
vast and varied. For example, prostate cancer remains an important
cause of cancer deaths and represents the second leading cancer of
males. It should also be noted that for purposes of disease and/or
fertility control, especially in humans, it may be desirable to use
the compositions to ablate pituitary gonadotrophs in conjunction
with other modes of treatment. For example, it is anticipated that
chronic administration of progestins and estrogens to females and
androgens to males might be necessary to prevent loss of secondary
sex characteristics, behavior and osteoporosis. Another area of
application in human medicine is treatment of endometriosis. This
condition, which produces painful growth of endometrial tissue in
the female peritoneum and pelvis also responds to inhibition of sex
steroid synthesis. Those skilled in this art will also appreciate
that the herein disclosed compounds could be used to partially
reduce sex-steroid secretions, and thus reduce or eliminate certain
hormone related behavior problems while retaining improved growth
stimulation.
[0112] The dose/time adjustments associated with the use of these
compounds can vary considerably; however, these compounds are
preferably administered by injection into a mammal in
concentrations of from about 0.1 to about 10 milligrams per
kilogram of the mammal's body weight. Sterilization may be
accomplished with as few as one injection; but multiple treatments
(e.g., based upon concentrations of from about 0.03 milligrams once
every 4 days to about 1 milligram per kilogram of body weight for
20 days) are alternative sterilization schemes. Furthermore, as
sterilization agents, the compositions can be used before or after
puberty.
[0113] The present invention will be further understood by
reference to the following non-limiting examples.
[0114] The following examples show that toxin could be encapsulated
in nanoparticles, and that nanoparticles labeled with a targeting
molecule could be targeted preferentially to target cells.
EXAMPLE 1
Conjugation of a Nanoparticle with a Targeting Molecule
[0115] A system based on LHRH was used to obtain a nanoparticle
carrier with a targeted molecule bound to its surface.
[0116] Materials and Methods
[0117] Reagents. NeutrAvadin.TM. labeled microspheres (0.04 .mu.m,
polystyrene, yellow-green fluorescent 505/515) were purchased from
Molecular Probes (Eugene, Oreg.). All other chemicals including
[Biotinyl-Gln.sup.1]-LHRH were obtained from Sigma (St Louis,
Mo.).
[0118] The following procedure was used to prepare the
nanoparticle-LHRH complex, with all steps performed at room
temperature:
[0119] 1) 1 mg of [Biotinyl-Gln.sup.1]-LHRH was dissolved in 5 mL
of Dulbecco's Phosphate Buffered Saline (DPBS) without Mg.sup.+ and
Ca.sup.2+ ions to create a stock solution.
[0120] 2) The NeutrAvadin.TM. labeled microspheres were sonicated
at 100 g for 10 minutes before undergoing dialysis for 5 hours to
remove any possible chemical preservatives. The dialysis buffer was
a solution of 1 part DPBS, 1 part distilled water.
[0121] 3) To 0.4 mL of the NeutrAvadin.TM. labeled microspheres was
added 218 mL of B-LHRH stock solution and 218 mL of a 1% solution
of BSA in DPBS.
[0122] 4) Dialysis was performed over a 24-hour period to remove
any unbound reagents, using the same buffer as in part (2). The
buffer was exchanged every 4 hours.
[0123] The clarity of the resulting solution suggested that
particle-particle aggregation, a notorious problem with targeted
nanoparticles, did not occur, i.e. that the nanoparticles remained
in solution at a submicron size.
EXAMPLE 2
Targeting of Nanoparticle Complex to Selected Cells
[0124] The following showed that nanoparticles labeled with LHRH
could be targeted to cells expressing LHRH receptor.
[0125] Materials and Methods
[0126] Reagents. Alpha minimum essential medium was purchased from
JRH Biosciences (Lenexa, Kans.). Penicillin-Streptomycin solution
was obtained from ATCC (Manassas, Va.). All other chemicals were
purchased from Sigma (St Louis, Mo.).
[0127] Cell Culture and Preparation. Rattus norvegicus (rat)
anterior pituitary cells, designation RC-4B/C, were purchased from
ATCC (Manassas, Va.). The cells were incubated in a 37.degree. C.,
5% CO.sub.2 atmosphere in a medium comprising Dulbecco's modified
Eagle's medium containing 2.2 g/L sodium bicarbonate, 4 mM
L-glutamine, and 4.5 g/L glucose, 45%; alpha minimum essential
medium with 1 g/L glucose, 45%; supplemented with 15 mM HEPES, 0.2
mg/mL bovine serum albumin, 2.5 ng/ml epidermal growth factor;
dialyzed heat-inactivated* fetal bovine serum, 10%. The medium was
supplemented with 50 units/mL penicillin and 0.05 mg/mL
streptomycin. *FBS was heat-inactivated using the ATCC-recommended
standard procedure.
[0128] Following ATCC guidelines, the complete growth medium was
refreshed every 2-3 days and subculture was performed every 5-6
days. The following procedure was used:
[0129] 1) All medium was discarded and a 37.degree. C. solution of
0.05% trypsin, 0.02% EDTA, 0.1% glucose was added to the cell
culture vessel at a volume of 0.1 mL per cm.sup.2 of cell
coverage.
[0130] 2) The cells were kept at 37.degree. C. for 7-10 minutes,
during which time they detached from the surface of the culture
vessel.
[0131] 3) An equal volume of complete growth medium was added to
the culture vessel before the cell suspension was transferred to a
sterile tube and pelleted through centrifugation at 100 g for 10
minutes.
[0132] 4) The pellet was resuspended in fresh 37.degree. C. growth
medium and placed into sterile culture vessels such that a
subculture ratio of 1:2 or 1:3 was achieved.
[0133] Endocytosis.
[0134] Experiments then were used to establish that targeted cells
can internalize a nanoparticle-targeting molecule conjugate with
greater efficiency than they internalize an unconjugated
nanoparticle of the same physical properties. The experimental
groups were as follows: The test nanoparticle/targeting molecule
complex was a conjugate of NeutrAvadin.TM. labeled microspheres
with [Biotinyl-Gln.sup.1]-LHRH, prepared as described in Example 1
above. The positive control group comprised carboxylate-modified
microspheres (0.04 .mu.m, polystyrene, yellow-green fluorescent
505/515) obtained from Molecular Probes (Eugene, Oreg.). The
negative control group comprised cells to which no particles had
been added; this group was used to determine cell autofluorescence.
After incubating equal concentrations of conjugate or carboxylated
nanoparticles with separate volumes of cell suspension (see
procedure below), the amount of fluorescence associated with the
cells was recorded using a flow cytometer.
[0135] Procedure
[0136] The procedure was identical for all three experimental
groups as described above (test, positive control, negative
control). Henceforth groups 1 and 2 are referred to as `particles`
14 hours in advance of flow cytometry:
[0137] 1) Cells were trypsinized and centrifuged according to the
subculturing procedure described above.
[0138] 2) Each pellet was resuspended in 2 mL preheated
calcium-free medium of the following composition: minimum essential
medium, Spinner Modification, containing 1 g/L glucose, 100%;
supplemented with 15 mM HEPES and 0.2 mg/mL bovine serum
albumin
[0139] 3) Cell suspension underwent constant gentle mixing at room
temperature for 12 hours.
[0140] 2 hours prior to flow cytometry:
[0141] 4) Cells were heated to 37.0.degree. C. (in the event of
overnight sedimentation, gentle resuspension was first
performed)
[0142] 5) After sonication (specification of sonication as
described in Example 1), particles were incubated with cells at a
concentration on the order of 100 particles/cell.
[0143] 6) After rapid agitation to ensure thorough mixing, the cell
suspensions were left to incubate with the particles at
37.0.degree. C. for 10 minutes.
[0144] 7) The cells were rapidly cooled before centrifugation and
resuspension of the resulting pellet (as described in subculturing
procedure) in 1 mL of calcium-free medium at 4.degree. C.
[0145] 8) Propidium Iodide was added to the cell suspensions at a
concentration of 50 .mu.l/mL
[0146] 9) The cells were kept at 4.degree. C. for a further 45
minutes prior to performing flow cytometry.
[0147] Results
[0148] FIGS. 1A and 1B illustrate that the amount of fluorescence
associated with cells that had been incubated with the test
conjugate was significantly greater than that associated with cells
which had been incubated with the control particles. This result
supports the claim that a nanoparticle conjugated with a targeting
molecule undergoes receptor-mediated endocytosis at a greater rate
than does a bare nanoparticle.
EXAMPLE 3
Synthesis of a Nanoparticle System Containing a Toxin Payload
[0149] Similar nanoparticles can be made containing toxins using
the procedures described below:
[0150] A nanoparticle system carrying a toxin payload can be made
using the procedure as described in: Pautot, Frisken, Weitz
"Production of unilamellar vesicles using an inverted emulsion"
(submitted for publication). Using Pautot et al's technique,
streptavidin-coated lipids (DPPC, DSPC, and similar lipids) can be
used to manufacture liposomes. Moreover, by adapting Needham's drug
encapsulation technique (Needham D, Newhirst M W. The development
and testing of a new temperature-sensitive drug delivery system for
the treatment of solid tumors. Advanced Drug Delivery Reviews, 53
(3): 285-305 Dec. 31 2001), it is also possible to load these
vesicles with doxorubicin, a model small-molecule toxin currently
used in cancer therapy.
[0151] The liposomes can be prepared by exposing chloroformic
solution of various lipid mixtures to high vacuum and subsequently
hydrating the resulting lipid films (DSPC/CHOL) with pH 4 buffers,
and extruding them through polycarbonated filters, after a freezing
and thawing procedure. It is possible to use DPPC supplemented with
DSPC or cholesterol to increase encapsulation efficiency or
increase stability, etc. A transmembrane pH gradient is created by
adjusting the pH of the extravesicular medium to 7.5 by addition of
an alkalinization agent. The anticancer drug doxorubicin is
subsequently entrapped by addition of the drug solution in small
aliquots to the vesicle solution, at an elevated temperature, to
allow drug accumulation inside the liposomes.
[0152] Once such streptavidin-coated and doxorubicin-loaded
liposomes are made, the procedure described in example 1 can be
repeated in order to attach [Biotinyl-Gln.sup.1]-LHRH to the
liposomes forming a liposome-LHRH complex. The protocol outlined in
example 2 can then be followed to study the efficacy of toxin
delivery to Rattus norvegicus anterior pituitary cells.
* * * * *